Limits...
Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Karki A, Humphrey SE, Steele RE, Hess DA, Taparowsky EJ, Konieczny SF - PLoS ONE (2015)

Bottom Line: Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses.We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells.The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, the Purdue Center for Cancer Research, and the Bindley Bioscience Center, Purdue University, West Lafayette, IN, 47907-2057, United States of America.

ABSTRACT
Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis-events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

Show MeSH

Related in: MedlinePlus

Mist1myc acinar cells exhibit extensive stromal infiltrates following AP induction.(A) Time course of iMist1myc induction and AP treatment. (B) H&E and IF analysis of iMist1myc pancreata post-AP. Arrows indicate remnants of acini structures. (C) iMist1 pancreata develop large increases in CD45+ immune infiltrates as well as VIMENTIN and SMA expressing stromal cells. (D) Immunoblot and RT-qPCR analysis of Vimentin and Sma levels in iMist1 samples post-AP. HSP90 was used as a loading control. Relative expression levels are indicated below the VIMENTIN panel, normalized to the corresponding HSP90 signal. Note that values were rounded to the nearest whole number to accomodate lane widths. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696804&req=5

pone.0145724.g006: Mist1myc acinar cells exhibit extensive stromal infiltrates following AP induction.(A) Time course of iMist1myc induction and AP treatment. (B) H&E and IF analysis of iMist1myc pancreata post-AP. Arrows indicate remnants of acini structures. (C) iMist1 pancreata develop large increases in CD45+ immune infiltrates as well as VIMENTIN and SMA expressing stromal cells. (D) Immunoblot and RT-qPCR analysis of Vimentin and Sma levels in iMist1 samples post-AP. HSP90 was used as a loading control. Relative expression levels are indicated below the VIMENTIN panel, normalized to the corresponding HSP90 signal. Note that values were rounded to the nearest whole number to accomodate lane widths. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Mentions: We next induced iMist1myc expression by treating Mist1CreERT/+/iMist1myc mice with Tam, followed by PBS (control) or caerulein to initiate an AP phenotype (Fig 6A). Surprisingly, instead of attenuating the AP response, Mist1CreERT/+/iMist1myc mice exhibited enhanced damage as early as 6h post-AP where extensive disruption of the exocrine pancreas occurred (Fig 6B and S6A Fig). By 2d-4d post-AP, the majority of acini structures were grossly altered with disorganized and distended lumens, a severe absence of eosinophilic zymogens, sustained elevated CLUSTERIN levels, and a large accumulation of infiltrating cells that included CD45+ immune cell populations (Fig 6B,6C and S6A–S6C Fig). During this period, the epithelial tissue mass was largely replaced by VIMENTIN+ and alpha-SMOOTH MUSCLE ACTIN (SMA)+ stromal cells (Fig 6C,6D and S6C Fig). The tissue also exhibited an increased islet density as the normal tissue mass that occupied space between available islets decreased, leaving the majority of the pancreas consisting of ductal, stromal and islet cells (Figs 6C and 7A). Protein immunoblots and RT-qPCR analyses revealed a typical AP damage profile with accumulation of CLUSTERIN protein, decreased expression of acinar gene products and increased expression of duct gene products over the 6h-2d post-AP period (Fig 7B–7D). However, by 7d-10d post-AP, despite reduced CLUSTERIN levels, ADM markers did not recover. Acinar genes (Amy, Cpa) remained suppressed while duct genes (K19, Sox9) continued to be expressed (Fig 7B–7D). Further analysis of these animals revealed a greatly decreased AMY+ acinar cell mass. At 2d and 4d post-AP, the vast majority of AMY+ acini co-expressed K19 in ADM structures (Fig 7D). Similarly, MIST1+ acinar cells were greatly decreased while stromal cells became more prominent within the exocrine tissue (Figs 6C, 7A, 7D and S6C Fig).


Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Karki A, Humphrey SE, Steele RE, Hess DA, Taparowsky EJ, Konieczny SF - PLoS ONE (2015)

Mist1myc acinar cells exhibit extensive stromal infiltrates following AP induction.(A) Time course of iMist1myc induction and AP treatment. (B) H&E and IF analysis of iMist1myc pancreata post-AP. Arrows indicate remnants of acini structures. (C) iMist1 pancreata develop large increases in CD45+ immune infiltrates as well as VIMENTIN and SMA expressing stromal cells. (D) Immunoblot and RT-qPCR analysis of Vimentin and Sma levels in iMist1 samples post-AP. HSP90 was used as a loading control. Relative expression levels are indicated below the VIMENTIN panel, normalized to the corresponding HSP90 signal. Note that values were rounded to the nearest whole number to accomodate lane widths. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696804&req=5

pone.0145724.g006: Mist1myc acinar cells exhibit extensive stromal infiltrates following AP induction.(A) Time course of iMist1myc induction and AP treatment. (B) H&E and IF analysis of iMist1myc pancreata post-AP. Arrows indicate remnants of acini structures. (C) iMist1 pancreata develop large increases in CD45+ immune infiltrates as well as VIMENTIN and SMA expressing stromal cells. (D) Immunoblot and RT-qPCR analysis of Vimentin and Sma levels in iMist1 samples post-AP. HSP90 was used as a loading control. Relative expression levels are indicated below the VIMENTIN panel, normalized to the corresponding HSP90 signal. Note that values were rounded to the nearest whole number to accomodate lane widths. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
Mentions: We next induced iMist1myc expression by treating Mist1CreERT/+/iMist1myc mice with Tam, followed by PBS (control) or caerulein to initiate an AP phenotype (Fig 6A). Surprisingly, instead of attenuating the AP response, Mist1CreERT/+/iMist1myc mice exhibited enhanced damage as early as 6h post-AP where extensive disruption of the exocrine pancreas occurred (Fig 6B and S6A Fig). By 2d-4d post-AP, the majority of acini structures were grossly altered with disorganized and distended lumens, a severe absence of eosinophilic zymogens, sustained elevated CLUSTERIN levels, and a large accumulation of infiltrating cells that included CD45+ immune cell populations (Fig 6B,6C and S6A–S6C Fig). During this period, the epithelial tissue mass was largely replaced by VIMENTIN+ and alpha-SMOOTH MUSCLE ACTIN (SMA)+ stromal cells (Fig 6C,6D and S6C Fig). The tissue also exhibited an increased islet density as the normal tissue mass that occupied space between available islets decreased, leaving the majority of the pancreas consisting of ductal, stromal and islet cells (Figs 6C and 7A). Protein immunoblots and RT-qPCR analyses revealed a typical AP damage profile with accumulation of CLUSTERIN protein, decreased expression of acinar gene products and increased expression of duct gene products over the 6h-2d post-AP period (Fig 7B–7D). However, by 7d-10d post-AP, despite reduced CLUSTERIN levels, ADM markers did not recover. Acinar genes (Amy, Cpa) remained suppressed while duct genes (K19, Sox9) continued to be expressed (Fig 7B–7D). Further analysis of these animals revealed a greatly decreased AMY+ acinar cell mass. At 2d and 4d post-AP, the vast majority of AMY+ acini co-expressed K19 in ADM structures (Fig 7D). Similarly, MIST1+ acinar cells were greatly decreased while stromal cells became more prominent within the exocrine tissue (Figs 6C, 7A, 7D and S6C Fig).

Bottom Line: Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses.We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells.The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, the Purdue Center for Cancer Research, and the Bindley Bioscience Center, Purdue University, West Lafayette, IN, 47907-2057, United States of America.

ABSTRACT
Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis-events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

Show MeSH
Related in: MedlinePlus